Permafrost and fire are important regulators of hydrochemistry and landscape structure in the discontinuous permafrost region of interior Alaska. We examined the influence of permafrost and a prescribed burn on concentrations of dissolved organic carbon (DOC), dissolved organic nitrogen (DON) and other solutes (, Ca2+, K+, Mg2+, Na+) in streams of an experimentally burned watershed and two reference watersheds with varying extents of permafrost in the Caribou–Poker Creeks Research Watershed in interior Alaska. The low-permafrost watershed has limited permafrost (3%), the high-permafrost watershed has extensive permafrost (53%), and the burn watershed has intermediate permafrost coverage (18%). A three end-member mixing model revealed fundamental hydrologic and chemical differences between watersheds due to the presence of permafrost. Stormflow in the low-permafrost watershed was dominated by precipitation and overland flow, whereas the high-permafrost watershed was dominated by flow through the active layer. In all watersheds, organic and groundwater flow paths controlled stream chemistry: DOC and DON increased with discharge (organic source) and base cations and (from weathering processes) decreased. Thawing of the active layer increased soil water storage in the high-permafrost watershed from July to September, and attenuated the hydrologic response and solute flux to the stream. The FROSTFIRE prescribed burn, initiated on 8 July 1999, elevated nitrate concentrations for a short period after the first post-fire storm on 25 July, but there was no increase after a second storm in September. During the July storm, nitrate export lagged behind the storm discharge peak, indicating a flushing of soluble nitrate that likely originated from burned soils. Copyright © 2006 John Wiley & Sons, Ltd.